Quantitation of Thyroglobulin in Serum Using SISCAPA and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

ABSTRACT

Accurate measurement of thyrogloblulin (Tg) at low concentrations is essential for recurrence-monitoring in patients who have been treated for papillary and follicular thyroid cancers. The immunoassays commonly employed by clinical laboratories to measure Tg are known to suffer interferences from thyroglobulin autoantibodies (TgAb).We describe a semiautomated stable isotope standards and capture by antipeptide antibodies (SISCAPA®) LC-MS/MS method for the accurate and precise measurement of Tg using 400 uL of serum. Following trypsin digestion of serum proteins in a 96-well plate format, a Tg-specific peptide is captured and concentrated using a monoclonal antibody bound to protein G-coated paramagnetic beads. Eighteen microliters of concentrate are injected into the LC-MS/MS system. Quantitation is performed against a 6-point linear calibration curve prepared in a blank matrix. The assay calibration range is 0.1-10 ng/mL, the range of clinical interest for recurrence detection. Total imprecision in clinical production has been observed to be 13.8% and 6.54% for in-house prepared control materials having Tg concentrations of 0.24 ng/mL and 0.94 ng/mL, respectively. Limit of quantitation was determined to be 0.1 ng/mL.